Fig. 7: Effect of a phosphorothioate bond on cleavage activity.
A Conformation of the phophodiester bond and of the two diastereoisomers of the phosphorothioate bonds. B Chromatograms of the DNA Pac FPLC of the top strand 13-mer with phosphodiester bonds (PDE) only (in blue) and of the 13-mer presenting a phosphorothioate bond (PTO) instead of the scissile PDE bond (in green). The black curve represent the NaCl gradient applied during the enantiomers separation. The first peak is the RP and the second the SP enantiomer. C Scheme of the enantiopure probe preparation. The oligonucleotides carrying a phosphorothioate bond, purchased from an industrial manufacturer, are a mixture of the two enantiomers. They were separated by HPLC, desalted, concentrated and then ligated to other oligonucleotides as indicated on the right to form the probe. D, E The control (Ctrl) represents the probe incubated without any enzyme. The probes obtained by ligation of the top- and bottom-strand 13-mers (PDE, PTO-RP, PTO-SP) were incubated with BsmI at 15 µg/mL at 65 °C for 10 min.
